During protein degradation through ATP-dependent proteases or during protein translocation across membranes such as those in mitochondria, proteins need to traverse narrow channels that are often too small to accommodate folded proteins, requiring unfolding. Somewhat surprisingly, the mechanisms of unfolding in vivo during translocation and degradation were found to resemble each other but differ from those of the global unfolding in vitro measured by solvent denaturation. In both cases, unfolding seems to be induced by physically pulling at the polypeptide chain, and the susceptibility of a protein to unravelling depends on the stability of the local structure that is adjacent to the targeting signal. Therefore, it seems attractive to speculate that the unfolding pathways involved in pore translocation resemble those sampled in in vitro single-molecule nanomechanical experiments. Moreover, our unanticipated findings showing that mechanically soft proteins cross the structurally large NPC faster than stiffer ones naturally lead us to speculate whether protein transport across other structurally wide pores such as the peroxisome can also be accelerated by the mechanical unfolding of the translocating protein cargo.
Combined, these independent observations across different pores call for reaching mechanistic understanding of the fleeting unfolding events encompassing protein translocation across biological pores of different natures, whether structurally stable or transiently formed, including peroxisomes, proteasomes and lysosomes.
Inspired by the mechanoselectivity of nuclear pores, in this PhD project we will use a combination of single-molecule nanomechanical experiments with single-cell optogenetic experiments (based on suitably engineered AsLOV2 light-excitable constructs that we will adapt to harbour specific recognition signals) to target a diverse range of cellular pores. We will explore whether a unifying mechanism that takes into account a protein’s mechanical stability and local structure as a master regulator of protein translocation can be drawn by systematically studying the translocation dynamics of a suite of proteins exhibiting a large range of mechanical activitiesacross biological pores of varying sizes and functions, including the peroxisome, the proteasome and the lysosome, in the context of chaperone-mediated autophagy.
The successful student will be trained in a variety of state-of-the-art techniques that encompass (i) single molecule force spectroscopy (AFM and magnetic tweezers), (ii) design of optogenetic constructs and (iii) light microscopy, complemented with bespoke training on molecular and cell biology.
The project will be conducted in the Garcia-Manyes laboratory, which is based both in the department of Physics of King’s College London (Strand Campus) and the Francis Crick Institute, and is funded by the Leverhulme Trust ‘Mechanics of Life’ Doctoral Scholarship Programme.
To be considered for the position candidates must apply via King’s Apply online application system.
Please follow the steps on the How to Apply section of the Mechanics of Life webpage carefully before submitting your application as applications that do not follow the correct application process may not be considered.
Once ready to submit, apply for the programme “Mechanics of Life Leverhulme Doctoral Scholarship Programme (MPhil / PhD)” quoting funding code MoL_DSP_2026 when doing so.